The present invention relates to antisense agents and methods for the treatment of disease states which are characterized by induction of p53. Such conditions include proliferative cell disorders, such as cancer, restenosis, and psoriasis, and hypoxic states induced by ischemic attacks such as stroke.
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The importance of p53 in cell proliferation is apparent from the observation that more than half of all human cancers display p53 mutations (Levine, 1997). An induction in p53 expression is observed in cells exposed to DNA alkylating agents, nitric oxide (Messmer, 1994), ionizing and ultraviolet irradiation (Lu and Lane, 1993), the restriction enzyme PvuII, which induces chromosome damage (Lu and Lane, 1993), and other agents capable of inducing DNA strand breaks (Nelson and Kastan, 1994). Ischemia/reperfusion (Raafat, 1997), models of epilepsy (Xiang, 1996), and hypoxia (Graeber, 1994) have also been demonstrated to induce p53. As shown herein and in previous studies (Rininger, 1997), p53 protein levels are also highly upregulated during the course of liver regeneration following partial hepatectomy, showing that damage of endogenous origin can also cause induction of p53 in vivo.
Roles for p53 have been postulated in cell cycle checkpoint activity, apoptosis, differentiation and DNA repair (Magnelli, 1997). Cell cycle checkpoints serve two important functions. One is to ensure that essential events in the cell cycle are completed before a subsequent event; the other is to provide more time for repair of damaged DNA before DNA replication and mitosis occur (Hartwell, 1994). The checkpoint activity of p53 occurs predominantly at the level of cells entering the S-phase (DNA synthesis) of the cell cycle (Kastan, 1992). G2-M checkpoint activity has also been reported for p53, due to its role as an upstream regulator of p21wafxe2x88x921. In the absence of either protein, DNA-damaged cells arrest in a G2-like state, but then undergo additional S phases without intervening normal mitoses (M phase), leading to nuclear abnormalities, e.g. polyploidy, and culminating in apoptosis (Waldman,
The invention provides, in one aspect, methods and compositions for treating a disease state characterized by p53 induction in a mammalian subject. The method comprises administering to the subject, in a suitable pharmaceutical carrier, a pharmaceutically effective amount of an antisense agent having the base sequence SEQ ID NO: 1, identified as 5xe2x80x2-TCA GTC TGA GTC AGG CCC-3, or the base sequence SEQ ID NO:2, identified as 5xe2x80x2-CCC TGC TCC CCC CTG GCT CC-3xe2x80x2, where the antisense agent is a morpholino oligonucleotide, a peptide nucleic acid, a 2xe2x80x2-O-allyl or 2xe2x80x2-O-alkyl modified oligonucleotide, a N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidate oligonucleotide, or a C-5-propyne pyrimidine-modified oligonucleotide. In a preferred embodiment, the antisense agent is a morpholino oligonucleotide, preferably having morpholino subunits joined by phosphorodiamidate backbone linkages. In another preferred embodiment, the antisense agent is a C-5-propyne pyrimidine-modified oligonucleotide.
The invention also includes compositions for use in treating such disease states. These compositions include antisense agents as described above, in a suitable pharmaceutical carrier.
In preferred applications of the method, the subject is a human subject. The disease state may be one which results from an ischemic or ischemic/reperfusive injury, such as stroke or the aftermath of organ transplant. Alternatively, the disease state may be cancer, in which case a preferred embodiment further comprises administering an agent effective to increase radical oxygen species at the cellular level. Such agents include radiosensitizing agents, ionizing radiation, a hyperbaric oxygen environment, and chemotherapeutic agents, such as certain anthracyclines or anthraquinones, which increase radical oxygen species at the cellular level.
For cancer treatment, the method may also include administering an agent effective to interfere with progression from the G2 phase to the M phase of the cell cycle. Such agents include phosphokinase C (PKC) inhibitors, bis(chloroethyl)nitrosourea (BCNU), pentoxifylline, silymarin, staurosporine, phenylahistin, paclitaxel, retinoic acid, flavopiridol, methyl-2,5-dihydrocinnamate, herboxidiene, 9-nitrocamptothecin, maitotoxin, apigenin, nocodazole, and colcemid.
In another aspect, the invention provides an oligonucleotide having a base sequence selected from the group consisting of SEQ ID NO:1, identified as 5xe2x80x2-TCA GTC TGA GTC AGG CCC-3xe2x80x2, and SEQ ID NO:2, identified as 5xe2x80x2-CCC TGC TCC CCC CTG GCT CC-3xe2x80x2, where the oligonucleotide is selected from the group consisting of a morpholino oligonucleotide, a peptide nucleic acid, a 2xe2x80x2-O-allyl or 2xe2x80x2-O-alkyl modified oligonucleotide, and a N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidate oligonucleotide. In one embodiment, the oligonucleotide is a morpholino oligonucleotide, preferably comprising morpholino subunits joined by phosphorodiamidate backbone linkages. The invention also provides an oligonucleotide having a base sequence selected from the group consisting of SEQ ID NO: 1, identified as 5xe2x80x2-TCA GTC TGA GTC AGG CCC-3xe2x80x2, and SEQ ID NO:2, identified as 5xe2x80x2-CCC TGC TCC CCC CTG GCT CC-3xe2x80x2, where the oligonucleotide is a C-5-propyne pyrimidine-modified oligonucleotide.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.